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Volatile Compounds from Androconial Organs of Danaine and Ithomiine Butterflies S. Schulz, W. Francke Universität Hamburg, Institut für Organische Chemie, Martin-Luther-King-Platz 6, D-2000 Hamburg 13, Bundesrepublik Deutschland

J. Edgar CSIRO, Division of Animal Health, P .O. Parkville, Vic. 3052, Australia

D. Schneider Max-Planck-Institut für Verhaltensphysiologie, D-8131 Seewiesen, Bundesrepublik Deutschland

Z. Naturforsch. 43c, 99-104 (1988); received July 29, 1987 Pyrrolizidine Alkaloids, Oxoisophorone, Bishomoterpenes, Butterfly-Pheromones, Danaines and Ithomiines, Buddleja Flowers

Pyrrolizidine alkaloid derivatives are present in the androconial (male scent) organs of Prittwit-zia hymenaea, Mechanitis isthmia veritabilis, Tithorea harmónia fúria (Lep., Ithomiinae), Amauris echeria and Euploea sylvester (Lep., Danainae). While the ithomiines contain the new pyrrolizidine alkaloid derivative methyl hydroxydanaidoate, the danaines contain the known derivatives danaidone and hydroxydanaidal. In addition, 2,2,6-trimethyl-2-cyclohexen-l,4-dione (oxoisophorone) and related terpenoids have been identified from Amauris, Euploea and Prittwit-zia as well as from the flowers of Buddleja davidii (Loganiaceae). Three new naturally occurring bishomoterpenes as well as mono- and sesquiterpenes and aromatic compounds add to the forma-tion of species specific blends in the scent organs of the butterflies.

Introduction

The closely related Danainae and Ithomiinae (Nymphalidae) are tropical butterflies known for their complex pheromone biology [1] and their mimicry. The male scent-producing organs (andro-conia) of Danainae consist of abdominal hairbrushes ("hairpencils") and alar pocket- or patch-like glands, while male Ithomiinae possess fringes of long hairs at the costal edge of the hindwings. Male and female danaine and ithomiine butterflies are known to ac-cumulate pyrrolizidine alkaloids (PAs) from plants [1], The males metabolize these to nitrogen contain-ing volatiles (dihydropyrrolizines) and to branched aliphatic acid derivatives which are secreted by the androconial organs [1], It appears that these com-pounds are important cues for sexual and/or social communication. The androconial organs are used differently in the Danainae and Ithomiinae. In most danaine genera (Amauris, Danaus, Ideopsis and Tirumala) male hairpencils are only expanded im-mediately prior to mating [1], however, in Euploea they are also displayed for longer periods during

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patrolling flights. In the case of the Ithomiinae, bio-assays by Haber [2] have provided evidence that the secretion of the alar fringes cause cross-attraction of ithomiines of both sexes and of different species and are thus responsible for the formation of multi-species assemblies (leks? [1]). Pliske, however, has obtained contrasting results which indicate inter- and intraspecies male repellency associated with the fringes of a number of ithomiine species and he has proposed that, in some species, components in the androconial secretions serve as male territorial-recognition pheromones and allomones [3],

Materials and Methods

The androconial organs were obtained from but-terflies collected in the field: 200 Amauris echeria (Stoll) (Danainae) from Kenya, East Africa, 15 Eu-ploea sylvester (F.) (Danainae) from Sri Lanka and 50 Prittwitzia hymenaea (Prittwitz) (Ithomiinae) from North-Argentine. Danaine hairpencils were artifi-cially protruded and excised, ithomiine fringes were cut-off with scissors. All organs were stored in pen-tane (Merck, Uvasol). These extracts were concen-trated to ca. 30 and analyzed by GC and GC/MS without preseparation of high boiling lipids. In addi-tion, methylene chloride extracts of the costal fringes

100 S. Schulz et al. • Volatile Compounds from Androconial Organs of Butterflies 100

of 150 male Mechanitis isthmia veritabilis (Butler) (Ithomiinae) and 15 Tithorea harmónia (Cramer) (Ithomiinae) from Venezuela were analyzed. Flow-ers of Buddleja davidii Franch. (Loganiaceae) were collected at different locations near Hamburg (FRG) and aerated in a modified closed loop stripping sys-tem apparatus [4] equipped with a 1.5 mg charcoal filter as a trap. After 5 h the filter was extracted 3 times with 10 |il of carbondisulfide. The crude extract was submitted to GC and GC/MS. Gas chro-matographic separations were carried out on a 60 m DB-5-CB capillary column programmed from 60 -280 °C at a rate of 3 °C/min, a 50 m WG-11 fused-silica column programmed from 60—220 °C at a rate of 3 °C/min and on a 1 m x 2 mm, glass-lined stain-less steel, 1% SE-30 packed column programmed from 7 0 - 2 2 0 °C at a rate of 6 °C/min. Mass spectra (70 eV) were obtained with a Varian MAT 311 A instrument connected to a Carlo-Erba 2150 gas Chromatograph and on a Varian M A T 111 A mass spectrometer linked to a Varian 1400 gas Chromato-graph. Identification of natural compounds base on comparison of mass spectra and retention times with those of synthetic samples.

2,6,6-Trimethyl-2-cyclohexen-l,4-dione (oxoiso-phorone) was prepared from commercially available isophorone [5]. Reduction of oxoisophorone with lithiumaluminiumhydride gave 4-hydroxy-2,6,6-tri-methyl-2-cyclohexenone and 4-hydroxy-3,5,5-tri-methyl-2-cyclohexenone which could be separated by preparative GC on SE-30. 2,2,6-Trimethylcyclo-hexan-l,4-dione was formed by base catalyzed rearrangement of 3,4-epoxy-3,5,5-trimethyl-2-cyclo-hexanone (ß-phoronoxide) [6]. (E)- and (Z)-8-Hy-droxy-4,8-dimethyl-4,9-decadienal were synthesized from nerolidol according to known methods [7]; the corresponding dihydroxy compound and the respec-tive hydroxy acid were obtained by reduction or oxi-dation respectively. Oxidation of the allylic alcohol, heliotridine, to the corresponding carboxylic acid methyl ester was achieved by Corey's method [8]. Further oxidation by manganese dioxide yielded 7-carbomethoxy-l ,2-dihydro-(3 H)-pyrrolizin-l-ol (methyl hydroxydanaidoate).

Results and Discussion

The androconial organs of the butterfly species in-vestigated here emit bouquets of volatiles composed of acetogenins, aromatic compounds, a variety of

terpenes, norterpenoids, bishomoterpenes and alka-loids. The compounds identified are compiled in Table I, chemical structures are shown in the formula scheme. Although the species investigated live on different continents their secretions were found to share closely related compounds obviously forming species specific compositions. Methyl hydroxy-danaidoate (XV), (£>4,8-dimethyl-4,9-decadien-1,8-diol (Xa), (£)-8-hydroxy-4,8-dimethyl-4,9-deca-dienal (Xb), and (£)-8-hydroxy-4,8-dimethyl-4,9-decadienoic acid (Xc) represent new natural prod-ucts, while the following compounds are new insect volatiles: 2,2,6-trimethylcyclohexan-l,4-dione (II), 4-hydroxy-2,6,6-trimethyl-2-cyclohexenone (V), 4-hydroxy-3,5,5-trimethyl-2-cyclohexenone (VI), 2-ethenyl-5-(l-hydroxy-l-methylethyl)-2-methyltetra-hydrofurane (VIII) and 2-ethenyl-5-hydroxy-2,6,6-trimethyltetrahydropyrane (IX). Several derivatives of PAs were found in the androconial organs of the investigated species. Such compounds are typical for Danainae and Ithomiinae, the males of which gather PAs as adults and use them for defence and as pre-

I I II H m

M VITT

X Q CH2OH b: CH0 c: C0 2H

2 a

IX

R1,R2=CH2OH R1=CO2H,R2=CH2OH R1 ,R2=CO2H

M M

HO CHO QH C0 2Me

tó c r i >

YN X2 Formula scheme: Structures of identified compounds, see also text in Table I.

S. Schulz et al. • Volatile Compounds from Androconial Organs of Butterflies 101

Table I. Volatile compounds identified from Amauris echeria (A. ech., 10 replications), Euploea sylvester (E. syl., 5 replications) and Prittwitzia hymenaea (P. hym., 3 replications), x < 10 ng, xx = 10—200 ng, xxx > 200 ng per individual.

Compound

2-Hexenal 11-Dodecanolide Benzaldehyde Phenylacetaldehyde Vanilline Eugenol Methylsalicylate Anisol Oxoisophorone (I) 2,2,6-Trimethylcyclohexan-l,4-dione (II) ß-Phorone (III) Isophorone (IV) 4-Hydroxy-2,6,6-trimethyl-2-cyclohexenone (V) 4-Hydroxy-3,5,5-trimethyl-2-cyclohexenone (VI) Linalool (VII) 2-Ethenyl-5-(l-hydroxy-l-methylethyl)-2-methyltetra-hydrofurane (VIII) 2-Ethenyl-5-hydroxy-2,6,6-trimethyltetrahydropyrane (IX) (E, £ ) -a -Farnesene Nerolidol (£)-4,8-Dimethyl-4,9-decadien-l,8-diol (Xa) (E)-8-Hydroxy-4,8-dimethyl-4,9-decadienal (Xb) (E)-8-Hydroxy-4,8-dimethyl-4,9-decadienoic acid (Xc) 2-Hydroxy-2-( l-hydroxyethyl)-3-methyl-y-butyrolactone (XII) Danaidone (XIII) Hydroxydanaidal (XIV) Methylhydroxydanaidoate (XV)

cursors for the biosynthesis of pheromone compo-nents [1, 9, 10]. A possible degradation product of pyrrolizidine alkaloids, 2-hydroxy-2-(2-hydroxy-ethyl)-3-methyl-y-butyrolactone (XII), which was previously found in seven species of Ithomiinae from four different genera [11] proved to be present in rather large amounts also in P. hymenaea, along with traces of a diastereomer. In A. echeria danaidone (XIII) was previously identified [12]; we confirmed its presence and additionally found the related hydroxydanaidal (XIV). Both danaidone (XIII) and hydroxydanaidal (XIV) were also identified in hair-pencils of E. sylvester [13]. In the ithomiines, P. hy-menaea, M. isthmia veritabilis and T. harmónia furia, we found "methyl hydroxydanaidoate" (XV) in rel-atively large amounts. The mass spectrum of (XV) is shown in Fig. 1. The observed diagnostic ions derive from loss of water (m/z 163), loss of a methoxy group (m/z 150), loss of carbomethoxy (m/z 122) and sub-sequent loss of water (m/z 104). Several cyclo-hexanone derivatives were identified in A. echeria,

A. ech. E. syl. P. hym.

xx XX

X X

X X X X XXX

X XXX X XX X X X

XXX X XX

XXX

X X X

X XXX XXX XXX

XX XXX XXX

X XXX XX

E. sylvester and P. hymenaea: while oxoisophorone (I; 2,6,6-trimethyl-2-cyclohexene-l,4-dione) is a prominent compound in A. echeria and P. hy-menaea, one of its reduction products (VI) was found as a major component in P. hymenaea exclusively. In contrast, another reduction product of oxoisopho-rone, (V), represents a major component in E. syl-vester. In P. hymenaea we identified considerable quantities of the ketones (I) and (VI) as well as traces of eugenol and the ketones (II) and (V). Oxo-isophorone (I) and isophorone (IV) have been previ-ously identified as trace constituents of the defensive secretion of a grasshopper species (Romalea [14]), while isophorone and ß-phorone (III) have been de-scribed as volatile constituents of females of the bark beetle, Ips typographus [15].

In the course of our studies on volatile constituents of flowers we analyzed the blossoms of Buddleja davidii, an ornamental garden bush, introduced to Europe from China. Its aromatic flowers are out-standingly attractive to butterflies and many other

xx x

102 S. Schulz et al. • Volatile Compounds from Androconial Organs of Butterflies 102

100 - | % .

50 -

Ulli, iL illl|i. lll|lili Ii U X r J 50 100 150

Fig. 1. 70 eV Mass spectrum of 7-carbmethoxy-~~I l,2-dihydro-(3H)-pyrrolizin-l-ol (methyl hy-

m/z 200 droxydanaidoate).

insects. We have identified oxoisophorone (I) as one of the two main compounds along with traces of its hydrogénation product, (II). Recently, the same ter-penoid cyclohexenone derivatives were found in another Buddleja species, B. salvifolia [16]. It is sur-prising to find (I) and (II) both as odour components in the secretions of flowers and insects, particularly since the butterflies (at least originally) are not sym-patric with Buddleja. Oxoisophorone (I) is a wide-spread natural product of plants and is also known to be produced by microorganisms [17] along with the related cyclic ketones (II), (V) and (VI). (E,E)-a-Farnesene represents the second main component in the odour bouquet of Buddleja, which besides ben-zylalcohol and benzaldehyde contains the monoter-penes (E)- and (Z)-ocimene, a-pinene, geraniol and ß-cyclocitral. ( f . i^ -a-Farnesene is also found in A. echeria.

The new bishomoterpenes (X) form another inter-esting group of compounds identified from the hair-brushes of E. sylvester. Similar compounds, (XI), identified by Meinwald et al. and by Edgar [18] from hairpencil extracts of Danaus spp. have been inter-preted as fixatives for volatiles, particular important in promoting adherence of pheromone-transfer parti-cles. While the structures of the Danaus compounds are related to geraniol, the new compounds Xa—c are obviously derived from linalool (VII), which is also present in the hairpencils of E. sylvester [13]. The biological function of Xa—c remains to be investi-gated. Other related compounds, showing a vinylcar-binol structure, are linalool (VII) and nerolidol

found in E. sylvester, while the cyclic linalool deriva-tives (VIII) and (IX) are present in A. echeria.

The mass spectra of Xb and of linalool are given in Fig. 2. Similarities in the spectra are obvious; the ra/z 71 fragment arises from the vinylcarbinol part of the molecules. The EI spectrum of Xb does not show the parent ion ra/z 196 while ions ra/z 178 (M-18) and ra/z 160 (M-18-18) indicate two oxygen atoms in the molecule. The ion ra/z 134 (M-44-18) suggests the presence of an aldehyde function. In the alcohol Xa the ion ra/z 119 shifts to ra/z 121. In the acid Xc the parent ion ra/z 212 remains invisible, while the ions ra/z 194 (M-18) and 134 (M-60-18), along with ra/z 60 and ra/z 73, indicate a carboxylic acid function. In the lower mass region of the compounds X the spectra look very similar.

Knowledge of the chemistry and biology of male pheromones of the Danainae [19] and Ithomiinae is limited, and the chemical data reported here contrib-ute new information in this regard which may also be of interest from a taxonomic and phylogenetic point of view. The identification of danaidone (XIII) as a major component in the extracts of E. sylvester from Sri Lanka is remarkable, since E. sylvester from Aus-tralia was said to show only traces of danaidone while none was reported in a study of the subspecies from Taiwan (Formosa), although in both of these earlier studies hydroxydanaidal (XIV) was identified [13]. This apparent difference in the PA-derived hairpen-cil constituents of E. sylvester from different regions warrants confirmation since it could indicate a biochemical difference in the subspecies concerned

S. Schulz et al. • Volatile Compounds from Androconial Organs of Butterflies 103

100 n

50 -

100 -n

50 -

I ' I "Ti 1 J--1 I 1 I 1 I 1 I 1 1 150 m/z 200

h 150 m/z 200 (below).

Fig. 2. 70 eV Mass spectra of linalool (above) and (Z^-S-hydroxy-^S-dimethyM.Ç-decadienal

and be evidence of incipient or latent speciation. Al-ternatively, danaidone may not be a particularly im-portant component in the complex hairpencil odour bouquet of E. sylvester, and the variability in its con-centration may not have any influence on the ability of these subspecies to interbreed in nature. It should be noted that also with regard to other hairpencil constituents samples from Sri Lanka and Australia (possible subspecies) differed significantly.

The identification of hydroxydanaidal (XIV) on the hairpencils of A. echeria extends the number of danaine genera now known to secrete this wide-spread PA metabolite which is particularly promi-nent in species of the genus Euploea but is also found, usually with other dihydropyrrolizines, in Tirumala, Parantica, Ideopsis and Danaus [13]. Simi-larly, detection of the lactone XII in P. hymenaea

increases the number of ithomiine genera (now five) known to secrete this PA metabolite [11], further indicating its wide, and this far unique, occurrence in the Ithomiinae.

The presence of the new dihydropyrrolizine, meth-ylhydroxydanaidoate (XV), in three ithomiine species from different genera is of particular interest since, until now, dihydropyrrolizines have been found only in the subfamily Danainae, although Pliske has reported Ehrlich reagent-positive chemi-cals, which he believed to be dihydropyrrolizines, in ithomiine androconial extracts [3] and Brown has also suggested that dihydropyrrolizine metabolites of PAs are present in some ithomiine species [10].

With regard to the non-PA-derived compounds identified, it is tempting to speculate on their origin. As can be seen from the formula scheme, the com-

104 S. Schulz et al. • Volatile Compounds from Androconial Organs of Butterflies 104

p o u n d s I—IX share a t e rpeno id ca rbon ske le ton . Al-though it is known tha t some insects are able to synthesize t e rpenes de novo [20], it s eems reasonab le to suggest t ha t , similar to o the r insects , the bu t t e r -flies seques te r these c o m p o u n d s or thei r p recursors f r o m plants . The cyc lohexenones m a y well be der ived f r o m plant ca ro teno ids [16], and a romat i c c o m p o u n d s are also k n o w n to be widesp read plant cons t i tuents .

Acknowledgements

W e t h a n k Dr . M. B o p p r é for helpful suggest ions and for kindly providing extracts of Amauris echeria. D r . T . E . Pliske provided I thomiids f r o m V e n e z u e l a . D . S. is g ra te fu l to D r . J. R . More l lo , Pres ident of the A r g e n t i n e Na t . Park Sys tem, for permiss ion to work in Iguazú . Financial suppor t by the D e u t s c h e For-schungsgemeinschaf t and the Fonds der Chemischen Indus t r ie is grateful ly acknowledged .

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